Lysosomal hydrolases thus far purified contain oligosaccharide chains which are presumably involved in lysosomal translocation. In the case of glucocerebrosidase, we have shown that the three forms of the enzyme found in fibroblasts are reduced to a single form upon removal of the carbohydrate by endoglycosidase F. Similarly, the single higher molecular weight form isolated from placental tissue upon endoglycosidase F treatment apparently differs only by its carbohydrate content from the forms found in fibroblasts. The carbohydrate structure of the placental enzyme has been determined and the number of attachment sites was proposed to be four per molecule. All sites were suggested to be N-glycosylated asparagines. This is in good agreement with the four sites suggested by endoglycosidase H treatment of the high mannose form of glycocerebrosidase. The cDNA sequence for the gene for glucocerebrosidase, determined in our laboratory, identified five potential N-glycosylated asparagines. In order to better understand the processing of lysosomal enzymes and define the exact number of carbohydrate sites in glucocerebrosidase, we have determined the amino acid sequence of the glycopeptides isolated from glucocerebrosidase. We have identified four glycopeptides, three from tryptic digests and one from cyanogen bromide. In each peptide, only one N-glycosylated asparagine was present. The fifth site, potentially present in glucocerebrosidase, was found to be a free asparagine. All four sites of carbohydrate attachment are the typical Asn(CHO)-X-Thr/Ser sequence. The sequences determined are: Asn(CHO)-Ala-Thr, Asn(Cho)-His-Thr, Asn(CHO)-Phe-Ser and Asn(CHO)-Ser-Thr. All the carbohydrate sites are located in the amino-terminal half of the molecule and may well play a role in both transport to the lysosome and localization in the membrane. Glycopeptides from Alpha-galactosidase A, sphingomyelinase, and iduronidase are being studied.